Isolation and Characterization of a Weizmannia coagulans Bacteriophage Youna2 and Its Endolysin PlyYouna2

Weizmannia coagulans (formerly Bacillus coagulans) is Gram-positive, and spore-forming bacteria causing food spoilage, especially in acidic canned food products. To control W. coagulans, we isolated a bacteriophage Youna2 from a sewage sludge sample. Morphological analysis revealed that phage Youna2 belongs to the Siphoviridae family with a non-contractile and flexible tail. Youna2 has 52,903 bp double-stranded DNA containing 61 open reading frames. There are no lysogeny-related genes, suggesting that Youna2 is a virulent phage. plyYouna2, a putative endolysin gene was identified in the genome of Youna2 and predicted to be composed of a N-acetylmuramoyl-L-alanine amidase domain (PF01520) at the N-terminus and unknown function DUF5776 domain (PF19087) at the C-terminus. While phage Youna2 has a narrow host range, infecting only certain strains of W. coagulans, PlyYouna2 exhibited a broad antimicrobial spectrum beyond the Bacillus genus. Interestingly, PlyYouna2 can lyse Gram-negative bacteria such as Escherichia coli, Yersinia enterocolitica, Pseudomonas putida and Cronobacter sakazakii without other additives to destabilize bacterial outer membrane. To the best of our knowledge, Youna2 is the first W. coagulans-infecting phage and we speculate its endolysin PlyYouna2 can provide the basis for the development of a novel biocontrol agent against various foodborne pathogens.


Introduction
Bacteriophages (phages) are bacteria-infecting viruses and known as the most abundant biological entities on Earth [1].Phages have been considered as an alternative antibacterial agent by their distinguishable features such as high specificity to the target bacteria and harmlessness to humans [2,3].In food industry, phages can be an effective and inexpensive tool to fight against foodborne pathogens [4].The application of phages to various foods has been reported to be successful in preventing foodborne pathogen contamination in recent years and an increasing number of phage products have been commercialized for controlling important foodborne pathogens [4,5].Beside the phage itself, endolysins have been also focused as an alternative strategy to control pathogenic bacteria.Endolysins are phage-encoded enzymes that hydrolyze the peptidoglycan of host bacteria at the end of the phage replication cycle, liberating of progeny phage particles from within [6,7].Endolysin can be externally applied to lyse bacterial cells, especially Gram-positive bacteria due to the absence of an outer membrane in the cell wall [8].Consequently, endolysin can access the peptidoglycan and kill unwanted bacteria.In addition, endolysins present several important advantages as biocontrol agents: low chance of developing bacterial resistance, high specificity for target bacteria without disturbing other bacteria, and strong enzymatic activity to rapidly lyse bacterial cells [9,10].These features make endolysin a promising and potent antimicrobial candidate, particularly in the light of increasing bacterial drug resistance.
Bacillus species are Gram-positive and spore-forming bacteria and one of the bacterial genera with the highest prevalence in the food industry.Despite their numerous advantages in industry applications, they could also be problematic [11].In particular, Weizmannia coagulans (re-classified from Bacillus coagulans, [12]) is known to be one of the important food spoilage microorganisms, frequently found in the canned fruit and vegetable processing industry [11,13].W. coagulans causes flat sour spoilage, drastically acidifying food products, eventually leading to significant economic losses in the food industry [14,15].Previously, several methods for controlling W. coagulans have been reported including heat treatment in the range from 95-103°C and thermochemical treatment using essential oils [16].Also, butyric acid vanillyl ester or caprylic acid vanillyl ester showed antibacterial activity against W. coagulans [17].However, these control methods could negatively affect the quality and sensory properties of foods, necessitating alternative antibacterial tools that inhibit the growth of these bacteria.Moreover, as the rapid emergence of antibiotic-resistant bacteria has become a global concern, it becomes important to develop a new type of control agent.To address these issues, here we isolated a novel W. coagulansinfecting phage, Youna2 and characterized its endolysin PlyYouna2.The endolysin gene, plyYouna2 was cloned and overexpressed in Escherichia coli, and the purified endolysin was biochemically characterized.PlyYouna2 showed strong lytic activity against W. coagulans and a number of other Gram-positive and Gram-negative bacteria, suggesting that PlyYouna2 has a relatively broad antibacterial spectrum.The results demonstrate that PlyYouna2 can be a promising antimicrobial agent against various foodborne pathogens.

Bacterial Strains and Growth Conditions
Table 1 shows the list of bacterial strains used in this study.W. coagulans KACC 15983 was used to isolate and propagate the phage Youna2.All W. coagulans strains were grown in tryptic soy broth (TSB, BD Difco, USA) at 50°C, and other Gram-positive bacteria including Bacillus and Staphylococcus strains were grown in TSB at 37°C.Clostridium perfringens cells were grown at 30°C under anaerobic conditions in brain heart infusion medium (BHI, BD Difco).Unless otherwise noted, cells were grown in Luria-Bertani (LB, BD Difco) broth at 37°C.

Bacteriophage Isolation and Propagation
Bacteriophage Youna2 was isolated from the secondary sludge of Jungnang Water Recycle Center in Seoul, South Korea.The sludge samples were mixed with 2X TSB in a 1:1 ratio and the overnight culture of Weizmannia coagulans KACC 15983 was added to the mixture in the presence of 0.5 mM CaCl 2 and 0.5 mM MgCl 2 , followed by incubation at 50°C at 250 rpm overnight.Then, the culture was harvested, and the supernatant was filtered using a  2 The percentage of lytic activity was obtained by the turbidity reduction assay for 1 h; 1-30% +, 30-70% ++; 71-100% +++, 0% -. 0.45 uM filter.The presence of phage in the filtrate was confirmed by spotting 10 μl of tenfold serially diluted filtrates onto soft agar (TSB containing 0.4% agar) containing 100 μl of host W. coagulans culture and 0.5 mM CaCl 2 and 0.5 mM MgCl 2 .The plates were incubated overnight at 50°C, and the formation of plaques was monitored.Clear plaques were picked with a sterile tip and eluted in SM buffer (Sodium-Magnesium Sulfate buffer).The plaque isolation steps were repeated at least three times as described above.
For phage propagation, W. coagulans strain was incubated at 50°C with shaking at 250 ×g until it reached an OD 600 of 0.7.Subsequently, phages at a multiplicity of infection (MOI) of 0.05 were added with 0.5 mM CaCl 2 and 0.5 mM MgCl 2 followed by a 3-h incubation at 50°C.The phage stock solution was prepared with the propagated phages as previously described [18].

Morphological Analysis by TEM
The morphology of Youna2 was analyzed using Energy-Filtering Transmission Electron Microscope (EF-TEM) as previously described [18].Youna2 was identified and classified according to the guidelines of the International Committee on Taxonomy of Viruses [19].

Host Range Analysis
The bacterial strains used for host range analysis were listed in Table 1.Each bacterial culture was mixed with 5 ml of TSB or LB soft agar and overlaid on tryptic soy agar (TSA) or LB agar plates.Subsequently, 10-fold diluted bacteriophage Youna2 lysates (10 10 to 10 3 plaque-forming units (PFUs)/ml) were spotted onto the plates and incubated overnight.The infectivity of Youna2 was determined based on the appearance of the plaques: "+", clear single plaques observed; "-", no lysis nor growth inhibition.

Bacteriophage DNA Extraction
Bacteriophage genomic DNA was purified as previously described [20].

Full-Genome Sequencing
Genomic DNA of phage Youna2 was sequenced using the Illumina MiSeq system at Sanigen Inc., South Korea and the de novo assembly algorithm of CLC Genomics Workbench 10.0.1 was used for genome assembly.Open reading frames (ORFs) annotation was conducted using the Rapid Annotation using Subsystem Technology (RAST) pipeline, as described previously [21].This annotation was further complemented using BLASTP [22] and Interproscan.The genome map was created by CGview server [23] and the complete genome sequence of W. coagulans phage Youna2 was deposited in GenBank (Accession number OM293949).

Cloning, Expression, and Purification of PlyYouna2
The predicted endolysin gene (plyYouna2) was amplified from the genomic DNA of the bacteriophage Youna2 by polymerase chain reaction (PCR) using primers, fBamH_PlyYouna2 (5'-GCG GGA TCC ATG GCA AGT AAA CCA TTG TTG GTG ATT G-3') and rHind_PlyYouna2 (5'-GCG AAG CTT TTA CCT CCT CTT CAC TGT GAC GTA CTT AGG TG-3').The PCR product was cloned into pET-28a (Novagen, USA), which has an Nterminal hexahistidine (His)-tag sequence.Plasmid with a correct insert was transformed into competent E. coli BL21 RIPL (DE3).Cells were grown in 2X YT broth with 50 μg/ml kanamycin and chloramphenicol to an OD 600 to 0.6-0.8, and the expression of the recombinant PlyYouna2 was induced with 0.5 mM isopropyl-b-dthiogalactopyranoside (IPTG), followed by incubation for an additional 24 h at 18°C.Bacterial cells were resuspended in lysis buffer (50 mM Tris-HCl, 500 mM sodium chloride and 30% glycerol, pH 8.0) and disrupted by sonication (Branson Ultrasonics, China).After centrifugation at 21,000 ×g for 1 h, the supernatant was obtained and filtered with 0.22 μm filter.The filtered supernatant was mixed with nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Germany) and incubated with rocking at 4 o C for 1 h.After the flow-through was discarded, the resin was serially washed using lysis buffer containing 10 mM imidazole, followed by lysis buffer with 20 mM imidazole.The protein was eluted using an elution buffer (50 mM Tris-HCl, 500 mM sodium chloride, and 200 mM imidazole, pH 8.0).The buffer of the eluted PlyYouna2 was changed to the lysis buffer using Zeba Spin Desalting Columns (MWCO 7 kDa, Thermo Fisher Scientific, USA) and stored at -80°C until use.

Lytic Activity Assay
The lytic activity of PlyYouna2 was assessed by the turbidity assay [26].Briefly, exponentially grown W. coagulans KACC 10117 was harvested and resuspended in reaction buffer (20 mM Tris-HCl, pH 8.0).PlyYouna2 (final molar concentrations of 0.4, 0.8, 1.2, and 1.6 μM) were incubated with the prepared bacterial cells, the OD 600 values were monitored for 1 h at room temperature.To evaluate the susceptibility of the target bacterial cells to PlyYouna2 in various pHs, 364 nM of PlyYouna2 was mixed with W. coagulans KACC 10117 cell suspensions in Britton-Robinson universal buffer (0.04 M H3PO 4 , 0.04 M H 3 BO 3 , 0.04 M CH 3 COOH, and 0.2 M NaCl).The thermal stability of the endolysin was tested by the lysis assays after the enzyme itself was incubated for 15 min at different temperatures.The effect of NaCl on lytic activity was evaluated at various NaCl concentrations (0-800 mM) [26,27].The relative activities were calculated by dividing the ΔOD 600 of each condition by the ΔOD 600 of the condition that showed the maximal activity.

Isolation and Characterization of W. coagulans Phage Youna2
The W. coagulans phage Youna2 was newly isolated from sewage sludge samples.This phage formed clear plaques with a halo against W. coagulans KACC 15983, a bacterial host strain, implying that tail proteins of Youna2 may have depolymerase activity (Fig. 1A) [28].EF-TEM revealed that phage Youna2 possesses an icosahedral head with a diameter of 50 nm and a non-contractile and flexible tail with a length of approximately 200 nm, indicating that phage Youna2 belongs to the family Siphoviridae (Fig. 1B).We performed a plaque assay to determine the host range of phage Youna2.As shown in Table 1, Youna2 was only able to show infectivity against W. coagulans strains, showing plaques on the lawn of the 4 out of 5 W. coagualns strains tested.Other Bacillus strains, Gram-positive bacteria or Gram-negative bacteria exhibited resistance to the phage Youna2, indicating that the infectivity of Youna2 is highly specific to its host.This finding explains one of the important characteristics of the phage, host specificity.

Genome Aanalysis of Phage Youna2
Whole genome sequencing analysis revealed genomic features of phage Youna2 (Fig. 2).Youna2 is a doublestranded DNA (dsDNA) virus with 52,903-base-pairs.The average G + C content of the genome is 42.72%, and it contains 61 ORFs with no rRNA or tRNA identified.The majority of the predicted genes (38 ORFs) encode hypothetical proteins with unknown functions.We assume that Youna2 is a virulent phage based on the fact that no lysogeny-related genes such as excisionase, genome attachment site (attP), repressor, integrase and transposase were identified in the genome of Youna2 [29].In addition, no genes related to bacterial virulence were identified.BLASTN revealed that the whole nucleotide sequence of Youna2 shares similarity with only four Bacillus phages (BSTP12, BSTP10, BSTP8, and BSTP5), with 4% query coverage and 75% identity, suggesting that phage Youna2 is a novel phage.Among the ORFs, phage tail protein which is a Siphovirus-type is identified in the genome, supporting that Youna2 belongs to the family Siphoviridae.Also, Youna2 encodes a gene involved in the host cell lysis, which is annotated as N-acetylmuramoyl-L-alanine amidase.

Identification and Overexpression of Youna2 Phage Endolysin
As described above, an 819 bp-long endolysin gene was identified from the whole genome sequence of phage Youna2 and is designated as plyYouna2.Functional domain analysis using InterProScan database predicted that PlyYouna2 is composed of the two domains with an N-terminal N-acetylmuramoyl-L-alanine amidase (8-179 aa) and C-terminal domain of unknown function DUF5776 (205-267 aa) (Fig. 3A).BLASTP analysis showed that the amino acid sequence is highly homologous to several N-acetylmuramoyl-L-alanine amidases (E.C. 3.5.1.28),sharing ~60% identity but most of them are from Bacillus strains such as B. licheniformis, B. atrophaeus, and B. pumilus (Fig. 3B).Considering that these bacterial autolysins have previously shown to cut the amide bond that connects the peptide and sugar moieties of the peptidoglycan [12], we assume that PlyYouna2 may have the same enzymatic activity.We obtained the predicted PlyYouna2 3D structure using AlphaFold2, an advanced tool for protein structure prediction with high confidence [24].AlphaFold analysis showed that PlyYouna2 has two distinct domains with a linker in between (Fig. 3C).The C-terminus domain of PlyYouna2 forms six β-sheets and the N-terminus domain comprised five α-helices and six β-sheets.The plyYouna2 gene was overexpressed in E. coli with an N-terminal His 6 -tag.The purified PlyYouna2 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 4A).The turbidity assay showed the strong lytic activity of PlyYouna2 against W. coagulans KACC10117 in a concentration-dependent manner (Fig. 4B).When 1.6 μM Youna2 was incubated with the cell suspension, the turbidity (OD 600 ) dropped to the basal level of detection within 30 min.Consequently, the results demonstrate that PlyYouna2 is a phage endolysin that can lyse W. coagulans cells and could be successfully applied exogenously as an effective antibacterial agent.

Antibacterial Spectrum of PlyYouna2
The antimicrobial spectrum of PlyYouna2 was tested against 5 Weizmannia strains, 9 Bacillus strains and other Gram-positive and Gram-negative bacteria (Table 1).All tested Weizmannia strains and Bacillus strains, except for B. cereus were lysed by PlyYouna2.These results are consistent with the idea that endolysin has a broader antibacterial spectrum than phage since endolysin targets the peptidoglycan of the bacterial cell wall [9].Geobacillus stearothermophilus, Listeria innocua, and L. monocytogenes were also susceptible to PlyYouna2.Interestingly, PlyYouna2-mediated cell lysis against Gram-negative bacteria including E. coli, Pseudomonas putida, Cronobacter sakazakii, and Yersinia enterocolitica was observed without the addition of outer membrane permeabilizers.Since PlyYouna2 is highly positive in charge (pI = 9.78) in the buffer condition we tested, its positive charge seems to be responsible for its interaction with negatively charged bacterial membrane, followed by destabilizing the membrane and killing bacteria cells [30,31].Recently, a number of endolysins that naturally lyse Gram-negative bacteria have been reported despite the fact that endolysins generally cannot access the peptidoglycan of Gram-negative bacteria [32,33].In these cases, it has been suggested that the presence of polyhistidine tags or N-terminal transmembrane regions of endolysin could be responsible for the lysis of Gramnegative bacteria but the outer membrane permeation mechanisms of the endolysins are still unclear [32,34,35].

Effect of NaCl, pH, and Temperature on the Lytic Activity of PlyYouna2
The relative lytic activity of PlyYouna2 was evaluated at different concentrations of NaCl, ranging from 0 to 800 mM and the assay was performed against W. coagulans KACC 10117 cells for 20 min at room temperature (Fig. 5A).PlyYouna2 exhibited the maximal activity in the presence of 200-400 mM NaCl.At concentrations higher than 400 mM NaCl, PlyYouna2 gradually lost its activity as the concentrations increase.PlyYouna2 was active in the pH range of 7.0-10.0,with the highest activity at pH 10.0 (Fig. 5B).However, no lytic activity was observed at pH 11 or pH 12.The thermostability of PlyYouna2 was determined at various temperatures.While PlyYouna2 retained 80% of its lytic activity after 15 min incubation at 4-60 o C, the lytic activity of PlyYouna2 dramatically dropped when it was incubated at 70 o C or higher (Fig. 5C).
In conclusion, Youna2 is the first virulent bacteriophage that infects the food spoilage bacterium, W. coagulans.An endolysin gene, plyYouna2 was identified in the genome of Youna2 and characterized.PlyYouna2 showed a broad antimicrobial spectrum and can even lyse Gram-negative bacteria without any outer membrane permeabilizers.These results suggest that PlyYouna2 could be used as a promising biocontrol agent against various foodborne pathogens including Gram-negative as well as Gram-positive bacteria.
Bacillus coagulans) is Gram-positive, and spore-forming bacteria causing food spoilage, especially in acidic canned food products.To control W. coagulans, we isolated a bacteriophage Youna2 from a sewage sludge sample.Morphological analysis revealed that phage Youna2 belongs to the Siphoviridae family with a non-contractile and flexible tail.Youna2 has 52,903 bp double-stranded DNA containing 61 open reading frames.There are no lysogenyrelated genes, suggesting that Youna2 is a virulent phage.plyYouna2, a putative endolysin gene was identified in the genome of Youna2 and predicted to be composed of a N-acetylmuramoyl-L-alanine amidase domain (PF01520) at the N-terminus and unknown function DUF5776 domain (PF19087) at the C-terminus.While phage Youna2 has a narrow host range, infecting only certain strains of W. coagulans, PlyYouna2 exhibited a broad antimicrobial spectrum beyond the Bacillus genus.Interestingly, PlyYouna2 can lyse Gram-negative bacteria such as Escherichia coli, Yersinia enterocolitica, Pseudomonas putida and Cronobacter sakazakii without other additives to destabilize bacterial outer membrane.To the best of our knowledge, Youna2 is the first W. coagulans-infecting phage and we speculate its endolysin PlyYouna2 can provide the basis for the development of a novel biocontrol agent against various foodborne pathogens.August 2023  Vol.33  No. 8

Fig. 3 .
Fig. 3. Modular structure of W. coagulans phage endolysin PlyYouna2.(A) PlyYouna2 domain organization that shows the locations of the predicted N-terminal amidase domain and the predicted C-terminal DUF5776 domain.Numbers indicate residue positions.(B) Sequence alignment of PlyYouna2-related enzymes.N-acetylmuramoyl-L-alanine amidase from B. licheniformis, B. atrophaeus, and B. pumilus.Conserved and identical residues are shaded in gray (dark gray, >70% conserved; light gray, >40% conserved) and black, respectively.(C) Cartoon representation of the PlyYouna2 3D structure predicted by AlphaFold2, colored from the N-terminus (blue) to the C-terminus (red).The N-terminal Amidase_3 and Cterminal DUF5776 domains are indicated.

Fig. 4 .
Fig. 4. (A) Purified PlyYouna2 was loaded on SDS-PAGE gels.Lane M, protein size marker.(B) Lysis of W. coagulans KACC 10117 with four different concentrations of PlyYouna2.The graph is representative of three independent replicates.

Fig. 5 .
Fig. 5. Biochemical characterization of PlyYouna2.The effects of NaCl concentration (A), pH (B), and temperature (C) on the lytic activity of PlyYouna2 against W. coagulans KACC 10117 cells are shown.Each column represents the mean of triplicate experiments, and error bars indicate the standard deviation.